Genetic locus imparting a low anatabine trait in tobacco and methods of using

Abstract

Provided herein are genetic markers and a coding sequence associated with a low- or ultra-low anatabine trait in tobacco.

1 .- 31 . (canceled) 
     
     
         32 . A method for producing a tobacco seed, the method comprising:
 (a) crossing a first tobacco plant having an induced mutation in an endogenous gene, the endogenous gene encoding the amino acid sequence of SEQ ID NO: 8, and wherein the first tobacco plant exhibits reduced expression or activity of a quinolinate synthase protein as compared to a control tobacco plant lacking the induced mutation, with a second tobacco plant,
 to produce at least one progeny tobacco seed; and 
   (b) selecting at least one progeny tobacco seed from step (a) that comprises the induced mutation.   
     
     
         33 . The method of  claim 32 , wherein the second tobacco plant comprises an induced mutation in an endogenous gene, the endogenous gene encoding the amino acid sequence of SEQ ID NO: 8, and wherein the second tobacco plant exhibits reduced expression or activity of a quinolinate synthase protein as compared to a control tobacco plant lacking the induced mutation. 
     
     
         34 . The method of  claim 32 , wherein the induced mutation results in an amino acid substitution at a position corresponding to position 487 of SEQ ID NO: 8 or position 516 of SEQ ID NO: 8. 
     
     
         35 . The method of  claim 34 , wherein the amino acid substitution at a position corresponding to position 487 of SEQ ID NO: 8 is a cysteine to tyrosine substitution. 
     
     
         36 . The method of  claim 34 , wherein the amino acid substitution at a position corresponding to position 516 of SEQ ID NO: 8 is a valine to isoleucine substitution. 
     
     
         37 . The method of  claim 32 , wherein the endogenous gene comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         38 . The method of  claim 32 , wherein the endogenous gene comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         39 . The method of  claim 32 , wherein the first tobacco plant is selected from the group consisting of a Burley tobacco plant, a dark tobacco plant, a flue-cured tobacco plant, a Maryland tobacco plant, and an Oriental tobacco plant. 
     
     
         40 . The method of  claim 32 , wherein the second tobacco plant is selected from the group consisting of a Burley tobacco plant, a dark tobacco plant, a flue-cured tobacco plant, a Maryland tobacco plant, and an Oriental tobacco plant. 
     
     
         41 . The method of  claim 32 , wherein a tobacco plant grown from the at least one progeny tobacco seed selected in step (b) exhibits a reduced amount of at least one alkaloid as compared to leaf from the control tobacco plant. 
     
     
         42 . The method of  claim 41 , wherein the at least one alkaloid is anatabine. 
     
     
         43 . A method of making a tobacco plant, the method comprising:
 (a) inducing mutagenesis in at least one tobacco cell to produce at least one mutagenized tobacco cell;   (b) regenerating at least one tobacco plant from the at least one mutagenized tobacco cell, wherein the at least one tobacco plant comprises an induced mutation in an endogenous gene, the endogenous gene encoding the amino acid sequence of SEQ ID NO: 8, and wherein the at least one tobacco plant exhibits reduced expression or activity of a quinolinate synthase protein as compared to a control tobacco plant lacking the induced mutation.   
     
     
         44 . The method of  claim 43 , wherein a leaf from the at least one tobacco plant exhibits a reduced amount of at least one alkaloid as compared to leaf from the control tobacco plant. 
     
     
         45 . The method of  claim 44 , wherein the at least one alkaloid is anatabine. 
     
     
         46 . The method of  claim 43 , wherein a cured leaf from the at least one tobacco plant exhibits a reduced amount of at least one tobacco-specific nitrosamine (TSNA) as compared to a cured leaf from the control tobacco plant. 
     
     
         47 . The method of  claim 46 , wherein the at least one TSNA is N′-nitrosoanatabine. 
     
     
         48 . The method of  claim 43 , wherein the inducing mutagenesis of step (a) comprises the use of a chemical mutagen or ionizing radiation. 
     
     
         49 . The method of  claim 48 , wherein the chemical mutagen is selected from the group consisting of nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethyl methane sulfonate. 
     
     
         50 . The method of  claim 43 , wherein the at least one tobacco cell is of a tobacco type selected from the group consisting of a Burley type, a dark type, a flue-cured type, a Maryland type, and an Oriental type. 
     
     
         51 . The method of  claim 43 , wherein the at least one tobacco cell is from a tobacco variety selected from the group consisting of FC401, Red Russian, BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116, VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, and VA359.